Desktop Tower Defense is played on a map resembling an office desktop. The player must stop a set number of enemies, known in the genre as "creeps", from reaching a set point on the playing field. This is accomplished by building and upgrading towers that shoot at, damage and kill the enemy creeps before they are able to reach their objective. Unlike many other tower defense games, the path of the creeps themselves is not set; instead, the towers built determine the path of the creeps, who take the shortest path they can find to the exit. The game does not allow the player to make an exit completely inaccessible, but key strategies revolve around guiding creeps into lengthy, meandering corridors. The creeps come in waves at set intervals. Each wave of creeps has different characteristics, such as immunity to certain towers, extra resistance to certain towers, and the abilities to spawn new creeps, move faster than normal, or fly over the towers instead of following the maze. For each wave, the game rotates among the types of creeps. Several times during gameplay, boss waves will appear. These waves consists of just one or two large creeps each, that take many hits to defeat. The player begins with a set number of lives, and loses one for each creep that survives to reach its goal (the edge of the grid opposite its entry-point, on most courses). The game will end early in defeat if the player's lives reach zero, and it provides no way to regenerate lost lives. The game can be played in three difficulty levels: easy, medium, and hard. In addition, there are several challenge modes available for those seeking more from the game. Challenges range from "The 100", where the player must defeat 100 levels of successively difficult creeps, to "3K Fixed", where the player has to build a fixed set of towers (including the upgrades) with just 3000 gold. There are also various fun modes different in gameplay than the other, harder ones.
The voice when a player is asked to enter a score for the high score table is sampled from Ivor the Engine.